Uses of hydroxy and ether-containing oxyalkylene esters for treating metabolic conditions

ABSTRACT

This invention relates to compositions for and methods of treating, preventing or ameliorating cancer and other proliferative diseases as well as methods of inducing wound healing, treating cutaneous ulcers, treating gastrointestinal disorders, treating blood disorders such as anemias, immunomodulation, enhancing recombinant gene expression, treating insulin-dependent patients, treating cystic fibrosis patients, inhibiting telomerase activity, treating virus-associated tumors, especially EBV-associated tumors, augmenting expression of tumor suppressor genes, inducing tolerance to antigens, or treating, preventing or ameliorating protozoan infection or inhibiting histone deacetylase in cells. The compositions of the invention are to and the methods of the invention use hydroxy and ether-containing oxyalkylene esters.

This application is a divisional application of U.S. Ser. No.08/814,224, filed Mar. 11, 1997 now U.S. Pat. No. 6,043,389.

FIELD OF THE INVENTION

This invention relates to compositions for and methods of treating,preventing or ameliorating cancer and other proliferative diseases aswell as methods of inducing wound healing, treating cutaneous ulcers,treating gastrointestinal disorders, treating blood disorders such asanemias, immunomodulation, enhancing recombinant gene expression,treating insulin-dependent patients, treating cystic fibrosis patients,inhibiting telomerase activity, treating virus-associated tumors,especially EBV-associated tumors, modulating gene expression andparticularly augmenting expression of tumor suppressor genes, inducingtolerance to antigens or treating, preventing or ameliorating protozoaninfection or inhibiting histone deacetylase in cells. The compositionsof the invention are to and the methods of the invention use hydroxy andether-containing oxyalkylene esters.

BACKGROUND OF THE INVENTION

Butyric acid (BA) is a natural product. It is supplied to mammals fromtwo main sources: 1) the diet, mainly from dairy fat, and 2) from thebacterial fermentation of unabsorbed carbohydrates in the colon, whereit reaches mM concentrations (Cummings, Gut 22:763-779, 1982; Leder etal., Cell 5:319-322, 1975).

BA has been known for nearly the last three decades to be a potentdifferentiating and antiproliferative agent in a wide spectra ofneoplastic cells in vitro (Prasad, Life Sci. 27:1351-1358, 1980). Incancer cells, BA has been reported to induce cellular and biochemicalchanges, e.g., in cell morphology, enzyme activity, receptor expressionand cell-surface antigens (Nordenberg et al., Exp. Cell Res. 162:77-85,1986; Nordenberg et al., Br. J. Cancer 56:493-497, 1987; and Fishman etal., J. Biol. Chem. 254:4342-4344, 1979).

Although BA or its sodium salt (sodium butyrate, SB) has been thesubject of numerous studies, its mode of action is unclear. The mostspecific effect of butyric acid is inhibition of nuclear deacetylase(s),resulting in hyperacetylation of histones H3 and H4 (Riggs, et al.,Nature 263:462-464, 1977). Increased histone acetylation, followingtreatment with BA has been correlated with changes in transcriptionalactivity and the differentiated state of cells (Thorne et al., Eur. J.Biochem. 193:701-713, 1990). BA also exerts other nuclear actions,including modifications in the extent of phosphorylation (Boffa et al.,J. Biol. Chem. 256:9612-9621, 1981) and methylation (Haan et al., CancerRes. 46:713-716, 1986). Other cellular organelles, e.g., cytoskeletonand membrane composition and function, have been shown to be affected byBA (Bourgeade et al., J. Interferon Res. 1:323-332, 1981). Modulationsin the expression of oncogenes and suppressor genes by BA weredemonstrated in several cell types. Toscani et al., reported alterationsin c-myc, p53 thymidine kinase, c-fos and AP2 in 3T3 fibroblasts(Oncogene Res. 3:223-238, 1988). A decrease in the expression of c-mycand H-ras oncogenes in B16 melanoma and in c-myc in HL-60 promyelocyticleukemia was also reported (Prasad et al., Biochem. Cell Biol.68:1250-1255, 1992; and Rabizadeh et al., FEBS Lett. 328:225-229, 1993).

BA has been reported to induce apoptosis, i.e., programmed cell death.SB has been shown to produce apoptosis in vitro in human coloncarcinoma, leukemia and retinoblastoma cell lines (Bhatia et al., CellGrowth Diff. 6:937-944, 1995; Conway et al., Oncol. Res. 7:289-297,1993; Hague et al.; Int. J. Cancer 60:400-406, 1995). Apoptosis is thephysiological mechanism for the elimination of cells in a controlled andtimely manner. Organisms maintain a delicate balance between cellproliferation and cell death, which when disrupted can tip the balancebetween cancer, in the case of over accumulation of cells, anddegenerative diseases, in the case of premature cell losses. Hence,inhibition of apoptosis can contribute to tumor growth and promoteprogression of neoplastic conditions.

The promising in vitro antitumor effects of BA and BA salts led to theinitiation of clinical trials for the treatment of cancer patients withobserved minimal or transient efficacy [Novogrodsky et al., Cancer51:9-14, 1983; Rephaeli et al., Intl. J. Oncol. 4:1387-1391, 1994;Miller et al., Eur. J. Cancer Clin. Oncol. 23:1283-1287, 1987].

Clinical trials have been conducted for the treatment of β-globindisorders (e.g., β-thalassemia and sickle-cell anemia) using BA salts.The BA salts elevated expression of fetal hemoglobin (HbF), normallyrepressed in adults, and favorably modified the disease symptoms inthese patients (Stamatoyannopouos et al., Ann. Rev. Med. 43:497-521,1992). In this regard, arginine butyrate (AB) has been used in clinicaltrials with moderate efficacy (Perrine et al., N. Eng. J. Med.328:81-86, 1993; Sher et al., N. Eng. J. Med. 332:1606-1610, 1995). Thereported side effects of AB included hypokalemia, headache, nausea andvomiting in β-thalassemia and sickle-cell anemia patients.

Butyric acid derivatives with antitumor activity and immunomodulatoryproperties have been reported in U.S. Pat. No. 5,200,553 and by Nudelmanet al., 1992, J. Med. Chem. 35:687-694. The most active buryric acidprodrug reported in these references was pivaloyloxymethyl butyrate(AN-9). Similar compounds are reported for treating hemoglobinopathies(U.S. Pat. No. 5,569,675).

BA and/or its analogues have also been reported to increase theexpression of transfected DNA (Carstea et al., 1993, Biophys. Biochem.Res. Comm. 192:649; Cheng et al., 1995, Am. J. Physical 268:L615-L624)and to induce tumor-restricted gene expression by adenovirus vectors(Tang et al., 1994, Cancer Gene Therapy 1:15-20). Tributyrin has beenreported to enhance the expression of a reporter gene in primary andimmortalized cell lines (Smith et al., 1995, Biotechniques 18:852-835).

However, BA and its salts are normally metabolized rapidly and have veryshort half-lives in vivo, thus the achievement and maintenance ofeffective plasma concentrations are problems associated with BA and BAsalts, particularly for in vivo uses. BA and BA salts have requiredlarge doses to achieve even minimal therapeutic effects. Because of thehigh dosage, fluid overload and mild alkalosis may occur. Patientsreceiving BA eminate an unpleasant odor that is socially unacceptable.

While BA salts have been shown to increase HbF expression, and appear tohold therapeutic promise with low toxicity in cancer patients, theynevertheless have shown low potency in in vitro assays and clinicaltrials. There remains a need to identify compounds as effective or moreeffective than BA or BA salts as differentiating or anti-proliferatingagents for the treatment of cancers. Such compounds need to have higherpotency than BA without the problems associated with BA (such as badodor). This need can be addressed by therapeutic compounds that eitherdeliver BA to cells in a longer acting form or which have similaractivity as BA but a longer duration of effectiveness in vivo.

The compounds of this invention address these needs and are more potentthan BA or BA salts for treating of cancers and other proliferativediseases, for treating gastrointestinal disorders, for wound healing,for treating blood disorders such as thalassemia, sickle cell anemia andother anemias, for modulating an immune response, for enhancingrecombinant gene expression, for treating insulin-dependent patients,for treating cystic fibrosis patients, for inhibiting telomeraseactivity, for treating virus-associated tumors, especiallyEBV-associated tumors, for modulating gene expression and particularlyfor augmenting expression of a tumor suppressor gene, for inducingtolerance to an antigen, for treating, preventing or amelioratingprotozoan infection and for inhibiting histone deacetylase in cells. Forexample, one of the advantages of the compounds of the invention isincreased hydrophilicity and thus easier administration, especially forintravenous administration.

SUMMARY OF THE INVENTION

The present invention is directed to compounds represented by theformula

wherein

R is C₁ to C₁₀ alkyl, C₂ to C₁₀ alkenyl or C₂ to C₁₀ alkynyl, optionallysubstituted with hydroxy, alkoxy, halo, trifluoromethyl, aryl orheteroaryl;

R¹ and R² are independently H, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, or C₂-C₁₀alkynyl, any of which can be optionally substituted with halo, alkoxy,amino, trifluoromethyl, aryl or heteroaryl;

R³ is

(1) C₁-C₁₂ alkyl or C₂-C₁₀ alkenyl substituted with from one to tensubstituents selected from the group consisting of hydroxy, alkoxy,acyloxy, aroyloxy and aryloxy, and, optionally having one or more halogroups, wherein adjacent hydroxy groups, if present, can optionally bepresent as an acetonide,

(2) —(CH₂)_(n)—[O—(CH₂)_(p)]_(m)—OR⁴,

(3) —(CH₂)_(n)—PEG—OR⁴, or

(4) —(CH₂)_(n)—PPG—OR⁴;

R⁴ is C₁ to C₆ alkyl, C₂ to C₆ alkenyl, aryl, acyl, aroyl,alkoxycarbonyl, aminocarbonyl or

PEG is a polyethylene glycol moiety having an average molecular weightof 200-8000;

PPG is a polypropylene glycol moiety having an average molecular weightof 200-8000;

p is 2 to 4;

n is 1 to 5;

m is 1 to 6; and

pharmaceutically-acceptable salts thereof;

with the proviso that when R is alkyl, R³ can not bemonohydroxy-n-propyl.

The above compounds are referred to herein as “Formula I compounds.” Ina preferred embodiment, the compounds of the invention are the compoundsof Formula I, wherein R³ is —(CH₂)_(n)—[O—(CH₂)_(p)]_(m)—OR⁴ , p is 2 or3, n is 1 or 2 and m is 1, 2 or 3. In a more preferred embodiment thecompounds of the invention are 2-(2-methoxyethoxy)acetyloxymethylbutyrate (Compound C), and 2-[2-(methoxyethoxy)ethoxy]acetyloxymethylbutyrate (Compound D), 1-[2-(2-methoxyethoxy)acetyl]oxyethyl butyrateand 1-{2-[2-(2-methoxyethoxy)ethoxy]acetyl}oxyethyl butyrate.

Another embodiment of the present invention is drawn to pharmaceuticalcompositions comprising a therapeutically effective amount of a compoundof Formula I and a pharmaceutically effective carrier or diluent.

A further embodiment of the present invention is directed topharmaceutical compositions comprising a therapeutically effectiveamount of a combination of a compound of Formula I with otheranti-cancer or anti-neoplastic agents together with a pharmaceuticallyeffective carrier or diluent.

Yet another embodiment of the present invention is directed to a methodof treating preventing or ameliorating cancer and other proliferativedisorders using the compounds of Formula I. In a preferred embodiment,the compounds of the invention used in this and the other methods of theinvention are the same preferred and more preferred compounds of FormulaI.

The compounds of Formula I are particularly useful for methods oftreating, preventing or ameliorating the effects of cancer and otherproliferative disorders by acting as anti-proliferative ordifferentiating agents in subjects afflicted with such anomalies. Suchdisorders include but are not limited to leukemias, such as acutepromyelocytic leukemia, acute myeloid leukemia, and acute myelomonocyticleukemia; other myelodysplastic syndromes, multiple myeloma such as butnot limited to breast carcinomas, cervical cancers, melanomas, coloncancers, nasopharyngeal carcinoma, non-Hodgkins lymphoma (NHL), Kaposi'ssarcoma, ovarian cancers, pancreatic cancers, hepatocarcinomas, prostatecancers, squamous carcinomas, other dermatologic malignancies,teratocarcinomas, T-cell lymphomas, lung tumors, gliomas,neuroblastomas, peripheral neuroectodermal tumors, rhabdomyosarcomas,and prostate tumors and other solid tumors. It is also possible thatcompounds of Formula I have anti-proliferative effects on non-cancerouscells as well, and may be of use to treat benign tumors and otherproliferative disorders such as psoriasis. Preferred is the method fortreating or ameliorating leukemia, squamous cell carcinoma andneuroblastoma.

The invention is further directed to a method of treating blooddisorders by administering a therapeutically-effective amount of acompound of Formula I to a patient.

The blood disorders treatable in accordance with the invention include,but are not limited to, thalassemias, sickle cell anemias, infectiousanemias, aplastic anemias, hypoplastic and hypoproliferative anemias,sideroblastic anemias, myelophthisic anemias, antibody-mediated anemias,anemias due to chronic diseases and enzyme-deficiencies, and anemias dueto blood loss, radiation therapy and chemotherapy. In this regard, thesemethods can include increasing hemoglobin content in blood byadminstering a therapeutically-effective amount of a compound of FormulaI to a subject.

Another embodiment of the invention is directed to a method ofmodulating an immune response in a host by administering an amount of acompound of Formula I effective to modulate said immune response.

Modulation of the immune response includes enhancing cytokine secretion,inhibiting or delaying apoptosis in polymorphonuclear cells, enhancingpolymorphonuclear cell function by augmenting hematopoietic growthfactor secretion, inducing expression of cell surface antigens in tumorcells, and enhancing progenitor cell recovery after bone marrowtransplantation.

Another embodiment of the present invention is directed to methods oftreating, preventing or ameliorating cancer and other proliferativedisorders by administering a therapeutically-effective amount of acompound of Formula I to a subject suffering from such disorderstogether with a pharmaceutical agent (e.g., a known antiproliferative,differentiating or oncostatic agent) to thereby enhance the action ofthese agents.

The pharmaceutical agents of the invention for the above method include,but are not limited to, cytokines, interleukins, anti-cancer agents,chemotherapeutic agents, antibodies, conjugated antibodies, immunestimulants, antibiotics, hormone antagonists, and growth stimulants. Thecompounds of the invention can be administered prior to, after orconcurrently with any of the agents.

Yet another embodiment of the invention is directed to a method ofameliorating the effects of a cytotoxic agent which comprisesadministering a therapeutically-effective amount of a cytotoxic agentwith a compound of Formula I to a mammalian patient for a time and in anamount to induce growth arrest of rapidly-proliferating epithelial cellsof the patient and thereby protect those cells from the cytotoxiceffects of the agent. The cytotoxic agent can be a chemotherapeuticagent, an anticancer agent, or radiation therapy. Rapidly proliferatingepithelial cells are found in hair follicles, the gastrointestinal tractand the bladder, for example. Such cells include hair follicle cells, orintestinal cryt cells. Rapidly proliferating cells are also found in thebone marrow and include bone marrow stem cells. In accordance with theinvention the cytotoxic agent and the compound of Formula I can beadministered simultanously, or the cytotoxic agent can be administeredprior to or after the compound of the invention. Administration(simultaneously or separately) can be done systemically or topically asdetermined by the indication. In addition, when the cytotoxic agent isradiation therapy, the compounds of the invention can be administered toa cancer patient pre- or post-radiation therapy to treat or amelioratethe effects of cancer.

A still further embodiment of the invention is directed to a method ofinducing wound healing, treating cutaneous ulcers or treating agastrointestinal disorder by administering a therapeutically-effectiveamount of a compound of Formula I to a subject in need of suchtreatment. The cutaneous ulcers which can be treated in accordance withthe methods of the invention include leg and decubitus ulcers, stasisulcers, diabetic ulcers and atherosclerotic ulcers. With respect towound healing, the compounds are useful in treating abrasions,incisions, burns, and other wounds. Gastrointestinal disorders treatableby the methods of the invention include colitis, inflammatory boweldisease, Crohn's disease and ulcerative colitis.

A further embodiment of the invention relates to a method of enhancingrecombinant gene expression by treating a recombinant host cellcontaining an expression system for a mammalian gene product of interestwith an expression-enhancing amount of a compound of Formula I, whereinsaid gene product is encoded by a butyric acid-responsive gene. The hostcells can be mammalian cells, insect cells, yeast cells or bacterialcells and the correspondingly known expression systems for each of thesehost cells. The gene product can be any protein or peptide of interest,expression of which can be regulated or altered by butyric acid or abutyric acid salt. A butyric acid-responsive gene is a gene that has apromoter, enhancer element or other regulon that modulates expression ofthe gene under its control in response to butyric acid or a salt ofbutyric acid. For example, gene products contemplated for regulation inaccordance with the invention include but are not limited to tumorsuppressor genes (such as p53) and the y-globin chain of fetalhemoglobin.

Yet a further embodiment of the invention is directed to a method oftreating, preventing or ameliorating symptoms in insulin-dependentpatients by administering an amount of a compound of Formula I effectiveto enhance insulin expression.

Yet another embodiment of the invention relates to a method of treating,preventing or ameliorating symptoms in cystic fibrosis patients byadministering an amount of a compound of Formula I effective to enhancechloride channel expression.

Still another method of the invention is directed to a method ofinhibiting telomerase activity in cancer cells by administering atelomerase-inhibiting amount of a compound of Formula I to the cells,wherein the amount is effective to decrease the telomerase activity ofthe cells and thereby inhibit the malignant progression of the cells.This method can be applied to in vivo or in vitro cells.

Another embodiment of this invention is directed to a method oftreating, preventing or ameliorating virus-associated tumors by pre-,post or co-administering a therapeutically-effective amount of acompound of Formula I with a therapeutically-effective amount of anantiviral agent. Antiviral agents contemplated for use in the inventioninclude ganciclovir, acyclovir and famciclovir, and preferablyganciclovir. The virus-associated tumors which can be treated, preventedor ameliorated in accordance with the invention include, but are notlimited to, EBV-associated malignancy, Kaposi's sarcoma, AIDS-relatedlymphoma, hepatitis B-associated malignancy or hepatitis C associatedmalignancy. EBV-associated malignancies include nasopharyngeal carcinomaand non-Hodgkins' lymphoma and are preferred embodiments of theinvention.

Further still the invention provides a method of modulating geneexpression by treating a host or host cells with an amount of a compoundof Formula I effective to enhance, augment or repress the expression ofa gene of interest, preferably a butyric acid responsive gene.

When expression of the gene of interest is to be enhanced or augmented,the gene can encode a gene product which is or acts as a repressor ofanother gene, a tumor suppressor, an inducer of apoptosis or an inducerof differentiation. When expression of the gene of interest is to berepressed, the gene can encode a gene product which is or acts as anoncogene or an inhibitor of apoptosis. For example, the bcl-2 geneencodes an inhibitor of apoptosis.

More particularly, the invention is directed to a method of augmentinggene expression, especially of a tumor suppressor gene, a butyricacid-responsive gene or a fetal hemoglobin gene, by treating a host orhost cells with an expression-enhancing amount of a compound of FormulaI. Preferably the host is a cancer patient. This method of the inventionthus includes augmenting tumor suppressor gene expression in conjunctionwith ex vivo or in vivo gene therapy, i.e., the compound of theinvention can be co-administered to the host during administration ofgene therapy vectors or administration of the ex vivo transfected cells.Similarly, the compounds of the invention can be used to treat cellsduring the transfection step of ex vivo gene therapy. The hosts of themethod therefore include cancer patients or other patients under goinggene therapy. The host cells of the invention include hematopoieticcells such as stem cells and progenitor cells, e.g., or any other celltype used in ex vivo gene therapy.

Yet another embodiment of the invention is directed to a method ofinducing tolerance to an antigen which comprises administering atherapeutically-effective amount of compound of Formula I. Preferablythe antigen is a self-antigen. For example, the self antigen can beassociated with an autoimmune disease, such as systemic lupuserythromatosus, rheumatoid arthritis, multiple sclerosis or diabetes.Alternatively, tolerance can be induced to one or more antigens presenton a transplanted organ or cells.

Yet further the invention is directed to a method for treating,preventing, or ameliorating protozoan infection in a subject whichcomprises administering to said subject an effective amount of acompound of Formula I. The protozoan infections treatable inaccordancewith the invention include, but are not limited to, malaria,cryptosporidiosis, toxoplasmosis, or coccidiosis.

Still further the invention is directed to a method of inhibitinghistone deacetylase in cells which comprises administering an effectiveamount of a compound of Formula I to said cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic illustration showing the in vitro inhibition ofcellular growth (clonogenicity) by AB, Compound C and Compound D onestablished HL-60 and CFPAC cancer cell lines as a function ofconcentration (in mM).

FIG. 2 is a graphic illustration showing the in vitro inhibition ofcellular growth by Compound C, compound D and AN9 on immortalized humankeratinocytes as a function of concentration (in mM).

FIG. 3 is a graphic illustration showing the percentage of humanleukemic cells (HL-60) expressing the phenotypic differentiation markerCD11b for cells which had been treated with Compound C or AN9 for 6 daysConcentrations are in μM.

DETAILED DESCRIPTION OF THE INVENTION

The compounds herein described may have asymmetric centers. All chiral,diastereomeric, and racemic forms are included in the present invention.Many geometric isomers of olefins and the like can also be present inthe compounds described herein, and all such stable isomers arecontemplated in the present invention.

By “stable compound” or “stable structure” is meant herein a compoundthat is sufficiently robust to survive isolation to a useful degree ofpurity from a reaction mixture, and formulation into an efficacioustherapeutic agent.

As used herein, “alkyl” means both branched- and straight-chain,saturated aliphatic hydrocarbon groups having the specified number ofcarbon atoms. As used herein “lower alkyl” means an alkyl group having 1to 5 carbon atoms. As used herein, “alkenyl” means hydrocarbon chains ofeither a straight or branched configuration and one or more unsaturatedcarbon-carbon bonds, such as ethenyl, propenyl, and the like. “Loweralkenyl” is an alkenyl group having 2 to 6 carbon atoms. As used herein,“alkynyl” means hydrocarbon chains of either a straight or branchedconfiguration and one or more carbon-carbon triple bonds, such asethynyl, propynyl and the like. “Lower alkynyl” is an alkynyl grouphaving 2 to 6 carbon atoms. When the number of carbon atoms is notspecified, then alkyl, alkenyl and alkynyl means lower alkyl, loweralkenyl and lower alkynyl, respectively.

As used herein, “aryl” includes “aryl” and “substituted aryl.” Thus“aryl” of this invention means any stable 6- to 14-membered monocyclic,bicyclic or tricyclic ring, containing at least one aromatic carbonring, for example, phenyl, naphthyl, indanyl, tetrahydronaphthyl(tetralinyl) and the like. The presence of substitution on the arylgroup is optional, but when present, the substituents can be halo,alkyl, alkoxy, hydroxy, amino, cyano, nitro, trifluoromethyl, acylaminoor carbamoyl. When a substituted aryl group is present on R or R³ inFormula I, then the aryl subsituents do not include amino, acylamino orcarbamoyl.

As used herein, the term “heteroaryl” includes “heteroaryl” and“substituted heteroaryl.” Thus “heteroaryl” of this invention means astable 5- to 10-membered monocyclic or bicyclic heterocyclic ring whichis aromatic, and which consists of carbon atoms and from 1 to 3heteroatoms selected from the group consisting of N, O and S and whereinthe nitrogen may optionally be quaternized, and including any bicyclicgroup in which any of the above-defined heteroaryl rings is fused to abenzene ring. The heteroaryl ring may be attached to its pendant groupat any heteroatom or carbon atom which results in a stable structure.The presence of substitution on the heteroaryl group is optional and canbe on a carbon atom, a nitrogen atom or other heteroatom if theresulting compound is stable and all the valencies of the atoms havebeen satisfied. When present, the substituents of the substitutedheteroaryl groups are the same as for the substituted aryl groups andalso include alkylammonium salts when the substituent in an alkyl groupattached to the nitrogen atom of the heteroaryl ring. These quarternizedammonium salts include halides, hydrohalides, sulfates, methosulfates,methanesulfonates, toluenesulfonates, nitrates, phosphates, maleates,acetates, lactates or any other pharmaceutically acceptable salt.Examples of heteroaryl groups include, but are not limited to, pyridyl,pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl,tetrazolyl, benzofuranyl, benzothienyl, indolyl, indolenyl, quinolinyl,isoquinolinyl and benzimidazolyl.

When a heteroaryl group is present on R in Formula I, then theheterotoms can not be a nitrogen. Similarly, if this same heteroarylgroup has any substituents, the substituents do not include amino,acylamino or carbamoyl, i.e., there can not also be an amino-typenitrogen-containing substiuent.

The term “substituted”, as used herein, means that one or more hydrogenson the designated atom are replaced with a selection from the indicatedgroups, provided that the designated atom's normal valency is notexceeded, and that the substitution results in a stable compound.

The substituents of the invention include, as indicated, halo, hydroxy,alkyl, alkoxy, amino, cyano, nitro, trifluoromethyl, aryl, heteroaryl,acyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, acyloxy, aryloxy,acetonide and aroyl. These groups can be substituents for alkyl,alkenyl, alkynyl, aryl, and heteroaryl groups as indicated in accordancewith the invention. A “halo” group is a halogen, and includes fluoro,chloro, bromo and iodo groups. The term “alkoxy” refers to an alkylgroup having at least one oxygen substituent represented by R—O—. Thegroup “acyl” is represented by the formula R—C(O)— where R is alkyl.“Arylcarbonyl” and “alkoxycarbonyl” are similar to acyl except that theR is aryl or alkoxy, respectively.

As used herein, “PEG” is a polyethylene glycol moiety having the formula—(OCH₂CH₂)_(x), where x is that number of subunits required to give anaverage molecular weight ranging from about 200 to about 8000. As usedherein, “PPG” is a polypropylene glycol moiety having the formula—(OCH₂CH₂CH₂)_(x), where x is that number of subunits required to givean average molecular weight ranging from about 200 to about 8000.

As used herein, “therapeutically-effective amount” refers to that amountnecessary to administer to a host to achieve an anti-tumor effect; toinduce differentiation and/or inhibition of proliferation of malignantcancer cells, benign tumor cells or other proliferative cells; to aid inthe chemoprevention of cancer; to promote wound healing; to treat agastrointestinal disorder; to treat a blood disorder or increase thehemoglobin content of blood; to modulate an immune response; to enhancerecombinant gene expression; to modulate gene expression; to augmentexpression of tumor suppressor genes; to enhance insulin expression; toenhance chloride channel expression, to induce tolerance to an antigen;to treat, prevent or ameliorate protozoan infection; or to inhibithistone deacetylase in cells. Methods of determiningtherapeutically-effective amounts are well known.

When the therapeutic or effective amount of the compound is fortreating, preventing or ameliorating cancer or other proliferativedisorder, then that amount can be an amount effective to inhibit histonedeacetylase in the subject, patient or cancerous cells. Similarly, whenthe therapeutic or effective amount of the compound is for treating,preventing, or ameliorating protozoan infection then that amount can bean amount effective to inhibit protozoan histone deacetylase in thesubject, patient or cancerous cells.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the disclosed compounds that are modified by making acid salts.Examples include, but are not limited to, mineral or organic acid saltsof basic residues such as amines. Pharmaceutically acceptable saltsinclude, but are not limited to, hydrohalides, sulfates, methosulfates,methanesulfonates, toluenesulfonates, nitrates, phosphates, maleates,acetates, lactates and the like.

Pharmaceutically-acceptable salts of the compounds of the invention canbe prepared by reacting the free base forms of these compounds with astoichiometric or greater amount of the appropriate acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. The salts of the invention can also be prepared by ionexchange, for example. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, p. 1418, the disclosure of which is hereby incorporated byreference in its entirety.

The “pharmaceutical agents” for use in the methods of the inventionrelated to the coadministration of compounds of Formula II and FormulaIII, include but are not limited to anticancer agents as well asdifferentiating agents. For example, the pharmaceutical agent can be acytokine, an interleukin, an anti-cancer agent, a chemotherapeuticagent, an antibody, a conjugated antibody, an immune stimulant, anantibiotic, a hormone antagonist or a growth stimulant. Thepharmaceutical agent can also be a cytotoxic agent. Cytotoxic agentsinclude antiviral nucleoside antibiotics such as ganciclovir, acyclovir,and famciclovir. Cytotoxic agents can also include radiation therapy.

As used herein, the “chemotherapeutic agents” include but are notlimited to alkylating agents, purine and pyrimidine analogs, vinca andvinca-like alkaloids, etoposide and etoposide-like drugs,corticosteroids, nitrosoureas, antimetabolites, platinum-based cytotoxicdrugs, hormonal antagonists, anti-androgens and antiestrogens.

The “cytokines” for use herein include but are not limited tointerferon, preferably α, β or γ interferon, as well as IL-2, IL-3,G-CSF, GM-CSF and EPO.

As used herein, an “immune stimulant” is a substance such as C. parvumor sarcolectin which stimulates a humoral or cellular component of theimmune system.

The chemotherapeutic agents of the invention include but are not limitedto tamoxifen, doxorubicin, L-asparaginase, dacarbazine, amascrine,procarbazine, hexamethylmelamine, mitoxantrone and gemcitabine.

Synthetic Methods

The compounds of the present invention can generally be prepared by anymethod known in the art. For example, the compounds of the invention canbe made by reacting the acid RCOOH with a reagent of the formula

or by similar reactions between any of the appropriate acids and theappropriate alkyl halides in the presence of a base, where Y is aleaving group such as halogen, methanesulfonate or p-toluenesulfonateand R, R¹, R² and R³ are as defined herein. The above reagents arereadily prepared according to literature procedures, see for example,Nudelman et al., J. Med. Chem. 35:687-694, 1992, and Japanese patent07033709 (1995). The base can be a trialkylamine, pyridine, an alkalimetal carbonate or other suitable base. The reaction can be carried outin the presence or absence of an inert solvent. Suitable solventsinclude, for example, acetone, benzene, toluene, tetrahydrofuran, ethylacetate, acetonitrile, dimethylformamide, dimethyl sulfoxide,chloroform, dioxan or 1,2-dichloroethane.

The procedures outlined above can be improved by one skilled in the artby, for instance, changing the temperature, duration, stoichiometry orother parameters of the reactions. Any such changes are intended to fallwithin the scope of this invention.

Activity

The activities of the compounds of the invention can be measured usinggenerally-accepted techniques known to those skilled in the artconsistent with the activity of interest. For example, the activity ofcompounds useful as differentiating agents can be measured usingstandard methodology of the nitro-blue tetrazolium reduction assay(e.g., Rabizadeh et al., FEBS Lett. 328:225-229, 1993; Chomienne et al.,Leuk. Res. 10:631, 1986; and Breitman et al. in Methods for Serum-freeCulture of Neuronal and Lymphoid Cells, Alan R. Liss, NY, p. 215-236,1984 which are hereby incorporated by reference in their entirety) andas described below. This in vitro assay has been deemed to be predictiveand in fact correlative with in vivo efficacy (Castaigne et al., Blood76:1704-1709, 1990).

Another assay which is predictive of differentiating activity is themorphological examination for the presence of Auer rods and/or specificdifferentiation cell surface antigens in cells collected from treatmentgroups, as described in Chomienne et al., (Blood 76:1710-1717, 1990which is hereby incorporated by reference in its entirety) and asdescribed below.

The compounds of the present invention also have anti-proliferative andanti-tumor activity. The anti-proliferation activity of compounds of thepresent invention can be determined by methods generally known to thoseskilled in the art. Generally-accepted assays for measuring viabilityand anti-proliferative activity are the trypan blue exclusion test andincorporation of tritiated thymidine, also as described by Chomienne, etal., above, which is incorporated herein by reference.

Other assays which predict and correlate antitumor activity and in vivoefficacy are the human tumor colony forming assay described in Shoemakeret al., Can. Res. 45:2145-2153, 1985, and inhibition of telomeraseactivity as described by Hiyayama et al., J. Natl. Cancer Inst.87:895-908, 1995, which are both incorporated herein by reference intheir entirety. These assays are described in further detail below.

Cell Cultures

Human promyelocytic leukemia cells (HL-60), human pancreatic carcinomacells (PaCa-2) and human breast adenocarcinoma cells, pleural effusioncells (MCF-7) can be cultured as follows. Cells are grown in RPMI mediawith 10% FCS, supplemented with 2 mM glutamine and incubated at 37° C.in a humidified 5% CO₂ incubator. Alternatively, cells can be grown inany other appropriate growth medium and conditions which supports thegrowth of the cell line under investigation. Viability can be determinedby trypan blue exclusion. Cells are exposed to a test compound, culturesare harvested at various time points following treatment and stainedwith trypan blue.

Cellular Staining to Detect Differentiation

Lipid staining and/or immunochemical staining of casein can be used as amarker for cellular differentiation of breast cancer cells (Bacus etal., Md. Carcin. 3:350-362, 1990). Casein detection can be done byhistochemical staining of breast cancer cells using a human antibody tohuman casein as described by Cheung et al., J. Clin. Invest.75:1722-1728, which is incorporated by reference in its entirety.

Nitro-Blue Tetrazolium (NBT) Assay:

Cell differentiation of myeloid leukemia cells can be evaluated, forexample, by NBT reduction activity as follows. Cell cultures are grownin the presence of a test compound for the desired time period. Theculture medium is then brought to 0.1% NBT and the cells are stimulatedwith 400 mM of 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Afterincubation for 30 min at 37° C., the cells are examined microscopicallyby scoring at least 200 cells. The capacity for cells to reduce NBT isassessed as the percentage of cells containing intracellular reducedblack formazan deposits and corrected for viability.

Cell Surface Antigen Immunophenotyping

Cell surface antigen immunotyping can be conducted using dual-colorfluorescence of cells gated according to size. The expression of a panelof antigens from early myeloid (CD33) to late myeloid can be determinedas described in Warrell, Jr. et al., New Engl. J. Med. 324:1385-1392,1992, which is incorporated by reference herein in its entirety.

Apoptosis Evaluation

Apoptosis can be evaluated by DNA fragmentation, visible changes innuclear structure or immunocytochemical analysis of Bcl-2 expression.

DNA fragmentation can be monitored by the appearance of a DNA ladder onan agarose gel. For example, cellular DNA is isolated and analyzed bythe method of Martin et al., J. Immunol., 145:1859-1867, 1990 which isincorporated by reference herein in its entirety.

Changes in nuclear structure can be assessed, for example, by acridineorange staining method of Hare et al., J. Hist. Cyt., 34:215-220, 1986which is incorporated by reference herein in its entirety.

Immunological detection of Bcl-2 can be performed on untreated cells andcells treated with the test compound.

HL-60 cells are preferred but other cell lines capable of expressingBcl-2 can be used. Cytospins are prepared and the cells are fixed withethanol. Fixed cells are reacted overnight at 4° C. with the primarymonoclonal antibody, anti-Bcl-2 at a dilution of 1:50. Staining iscompleted to visualize antibody binding, for example, using Strep A-BUniversal Kit (Sigma) in accordance with the manufacturer'sinstructions. Identically-treated cells which received no primaryantibody can serve as a non-specific binding control. Commercial kitsare also available and can be used for detecting apoptosis, for example,Oncor's Apop Tag®.

Modulation of Gene Expression

The levels of expression from oncogene and tumor suppressor genes can beevaluated by routine methods known in the art such as Northern blottingof RNA, immunoblotting of protein and PCR amplification.

Mouse Cancer Model

Compounds can be examined for their ability to increase the life span ofanimals bearing B16 melanomas, Lewis lung carcinomas and myelomonocyticleukemias as described in Nudelman et al., J. Med. Chem. 35:687-694,1992, or Rephaeli et al., Int. J. Cancer 49:66-72, 1991, which areincorporated by reference herein in their entireties.

For example, the efficacy of compounds of the present invention in aleukemia model can be tested as follows: Balb/c mice are injected withWEHI cells and a test compound or control solution is administered thefollowing day. The life span of the treated animals is compared to thatof untreated animals.

The efficacy of compounds of the present invention on primary tumors canalso be tested with subcutaneously implanted lung carcinoma or B16melanoma by measuring the mass of the tumor at the site of implantationevery two weeks in control and treated animals.

The efficacy of compounds in xenografts can be determined by implantingthe human tumor cells subcutaneously into athymic mice. Human tumor celllines which can be used include, but are not limited to, prostatecarcinoma (human Pc-3 cells), pancreatic carcinoma (human Mia PaCacells), colon adenocarcinoma (human HCT-15 cells) and mammaryadenocarcinoma (human MX-1 cells). Treatment with control solution or atest compound of the invention begins, for example, when tumors areapproximately 100 mg. Anti-tumor activity is assessed by measuring thedelay in tumor growth, and/or tumor shrinking and/or increased survivalof the treated animals relative to control animals.

Telomerase Activity

High levels of telomerase activity is associated with the highproliferation rate found in cancer cells. Compounds which inhibittelomerase activity results in inhibition of cancer cell growth andde-differentiation. Commercially available telomerase assays can thus beused to assess the anticancer activities of compounds on cancer celllines.

Chemoprevention

The chemoprevention activity of the compounds of the invention can bedetermined in the two-stage mouse carcinogenesis model of Nishimo et al.(supra).

Assay of Compounds

Compounds of the invention, their salts or metabolites, can be measuredin a biological sample by any method known to those skilled in the artof pharmacology, clinical chemistry or the like. Such methods formeasuring these compounds are standard methods and include, but are notlimited to high performance liquid chromatography (HPLC), gaschromatography (GC), gas chromatography mass spectroscopy (GC-MS),radioimmunoassay (RIA), and others.

Dosage and Formulation

The compounds of the present invention can be administered to amammalian patient to treat cancer or in any other method of theinvention which involves treating a patient by any means that producescontact of the active agent with the agent's site of action in the bodyof the subject. Mammalian patients include humans and domestic animals.The compounds of the invention can be administered by any conventionalmeans available for use in conjunction with pharmaceuticals, either asindividual therapeutic agents or in a combination of therapeutic agents.The compounds can be administered alone, but are generally administeredwith a pharmaceutical carrier selected on the basis of the chosen routeof administration and standard pharmaceutical practice. Thepharmaceutical compositions of the invention may be adapted for oral,parenteral, transdermal, transmucosal, rectal or intranasaladministration, and may be in unit dosage form, as is well known tothose skilled in the pharmaceutical art. The term “parenteral” as usedherein includes subcutaneous, intravenous, intramuscular, orintrasternal injection or infusion techniques.

The appropriate dosage administered in any given case will, of course,vary depending upon known factors, such as the pharmacodynamiccharacteristics of the particular agent and its mode and route ofadministration; the age, general health, metabolism, weight of therecipient and other factors which influence response to the compound;the nature and extent of the symptoms; the kind of concurrent treatment;the frequency of treatment; and the effect desired. A daily dosage ofactive ingredient can be expected to be about 10 to 10,000 milligramsper meter² of body mass (mg/m²), with the preferred dose being 50-5,000mg/m² body mass.

Dosage forms (compositions suitable for administration) contain fromabout 1 mg to about 1 g of active ingredient per unit. In thesepharmaceutical compositions the active ingredient will ordinarily bepresent in an amount of about 0.5-95 by weight based on the total weightof the composition.

The active ingredient can be administered orally in solid or semi-soliddosage forms, such as for example hard or soft-gelatin capsules,tablets, and powders, or in liquid dosage forms, such as elixirs,syrups, disperse powders or granules, emulsions, and aqueous or oilysuspensions. It can also be administered parenterally, in sterile liquiddosage forms. Other dosage forms include transdermal administration viaa patch mechanism or ointment.

Compositions intended for oral use may be prepared according to anymethods known to the art for the manufacture of pharmaceuticalcompositions and such compositions may contain one or more agentsincluding sweetening agents, flavoring agents, coloring agents, andpreserving agents in order to provide a pharmaceutically elegant andpalatable preparation.

Tablets contain the active ingredient in admixture with non-toxicpharmaceutically acceptable excipients which are suitable for themanufacture of tablets. Such excipients may include, for example, inertdiluents, such as calcium phosphate, calcium carbonate, sodiumcarbonate, sodium phosphate, or lactose; granulating disintegratingagents, for example, maize starch or alginic acid; binding agents, suchas starch, gelatin, or acacia; and lubricating agents, for example,magnesium stearate, stearic acids or talc. Compressed tablets may beuncoated or may be sugar coated or film coated by known techniques tomask any unpleasant taste and protect the tablet from the atmosphere, orenteric coated for selective disintegration and adsorption in thegastrointestinal tract.

Hard gelatin capsules or liquid filled soft gelatin capsules contain theactive ingredient and inert powdered or liquid carriers, such as, butnot limited to calcium carbonate, calcium phosphate, kaolin, lactose,lecithin starch, cellulose derivatives, magnesium stearate, stearicacid, arachis oil, liquid paraffin, olive oil, pharmaceutically-acceptedsynthetic oils and other diluents suitable for the manufacture ofcapsules. Both tablets and capsules can be manufactured as sustainedrelease-products to provide for continuous release of medication over aperiod of hours.

Aqueous suspensions contain the active compound in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, e.g., sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose, sodium alginate,polyvinylpyrrolidone, gum tragacanth, and gum acacia; dispersing orwetting agents, such as a naturally occurring phosphatide, e.g.,lecithin, or condensation products of an alkylene oxide with fattyacids, for example of polyoxyethylene stearate, or a condensationproducts of ethylene oxide with long chain aliphatic alcohols, e.g.,heptadecaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol, e.g.,polyoxyethylene sorbitol monooleate, or a condensation product ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides, e.g., polyoxyethylene sorbitan monooleate. The aqueoussuspensions can also contain one or more preservatives, for exampleethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents, and one or more sweetening agents, such assucrose, saccharin, or sodium or calcium cyclamate.

Dispersable powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example, sweetening, flavoring, and coloringagents, can also be present.

Syrups and elixirs can be formulated with sweetening agents, such asglycerol, sorbitol or sucrose. Such formulations may also contain ademulcent, a preservative and flavoring and coloring agents.

The pharmaceutical compositions can be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueoussuspension. This suspension can be formulated according to the known artusing those suitable dispersing or wetting agents and suspending agentswhich have been mentioned above. The sterile injectable preparation canalso be a sterile injectable solution or suspension in a nontoxicparenterally-acceptable diluent or solvent, for example, as a solutionin 1,3-butanediol.

In general, water, a suitable oil, saline, aqueous dextrose (glucose),polysorbate and related sugar solutions, emulsions, such as Intralipid®(Cutter Laboratories, Inc., Berkley Calif.) and glycols such aspropylene glycol or polyethylene glycols are suitable carriers forparenteral solutions. Antioxidizing agents, such as but not limited tosodium bisulfite, sodium sulfite, or ascorbic acid, either alone orcombined, are suitable stabilizing agents. Also used can be citric acidand its salts and sodium EDTA. In addition, parenteral solutions cancontain preservatives, such as but not limited to benzalkonium chloride,methyl- or propyl-paraben, and chlorobutanol.

The pharmaceutical compositions of the present invention also includecompositions for delivery across cutaneous or mucosal epitheliaincluding transdermal, intranasal, sublingual, buccal, and rectaladministration. Such compositions may be part of a transdermal device,patch, topical formulation, gel, etc., with appropriate excipients.Thus, the compounds of the present invention can be compounded with apenetration-enhancing agent such as 1-n-dodecylazacyclopentan-2-one orthe other penetration-enhancing agents disclosed in U.S. Pat. Nos.3,991,203 and 4,122,170 which are hereby incorporated by reference intheir entirety to describe penetration-enhancing agents which can beincluded in the transdermal or intranasal compositions of thisinvention.

Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, Mack Publishing Company, a standard referencetext in this field, which is incorporated herein by reference in itsentirety.

Various modifications of the invention in addition to those shown anddescribed herein will be apparent to those skilled in the art from theforegoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

The foregoing disclosure includes all the information deemed essentialto enable those skilled in the art to practice the claimed invention.Because the cited patents or publications may provide further usefulinformation these cited materials are hereby incorporated by referencein their entirety.

EXAMPLE 1 Synthesis of 2-(2-Methoxyethoxy)acetyloxymethyl Butyrate

2-(2-Methoxyethoxy)acetyloxymethyl butyrate (Compound C) has thefollowing structure

and was synthesized as follows. To a solution of chloromethyl butyrate(682 mg, 5 mmol) in acetone (10 mL), 2-(2-methoxyethoxy)acetic acid (570mg, 5 mmol) was added, followed by the dropwise addition oftriethylamine (0.7 mL, 0.5 g, 1 eq). The mixture was heated at 40° C.for 20 h, until TLC (ethyl acetate:hexane 2:1, ninhydrin) indicated thetotal consumption of the starting materials. The precipitate wasfiltered and washed with acetone. The filtrate was evaporated and theresidue (1.08 g) was chromatographed on silica gel (ethylacetate:hexane, 2:1) to give the product as an oil, 520 mg (44% yield).

EXAMPLE 2 Synthesis of 2-[2-(Methoxyethoxy)ethoxy]acetyloxymethylButyrate and Additional Compounds

2-[2-(Methoxyethoxy)ethoxy]acetyloxymethyl butyrate (Compound D) has thefollowing structure

and was synthesized as follows. To a solution of chloromethyl butyrate(682 mg, 5 mmol) in acetone (10 mL), 2-[2-(methoxyethoxy)ethoxy]aceticacid (890 mg, 5 mmol), was added, followed by the dropwise addition oftriethylamine (0.7 mL, 0.5 g, 1 eq). The mixture was heated at 40° C.for 20 h, until TLC (ethyl acetate:hexane 2:1, ninhydrin) indicatedcomplete consumption of the starting materials. The precipitate wasfiltered and washed with acetone. The filtrate was evaporated and theresidue (1.35 g) was chromatographed on silica gel (ethylacetate:hexane, 2:1), to give the product as an oil (390 mg, 28i yield).

Additional compounds of the invention are provided in Table I. Thesecompounds are those of Formula I having the designated groups. Thesecompounds can be synthesized in a manner analogous to Examples 1 and 2or as provided in the Detailed Description of the Invention.

TABLE 1 Additional Compounds R R¹ R² R³ n-C₃H₇ CH₃ H —CH₂OCH₂CH₂OCH₃n-C₃H₇ C₃H₇ H —CH₂OCH₂CH₂OCH₃ n-C₃H₇ CH₃ H —CH₂—(OCH₂CH₂)₂—OCH₃ n-C₃H₇CH₃ CH₃ —CH₂—(OCH₂CH₂)₂—OCH₃ n-C₃H₇ H H —CH₂-PEG₂₀₀-OC₂H₅ n-C₃H₇ CH₃ H

n-C₃H₇ CH₃ H

CH₂═CHCH₂— CH₃ H CH₂—(OCH₂CH₂)₆OC₂H₅ ClCH₂CH₂CH₂— H H

C₂H₅ H

H H

CH₂═CHCH₂ H

C₆H₅CH₂ C₃H₇ H

EXAMPLE 3 Clonogenicity of Established Tumor Cell Lines

Inhibition of tumor growth was tested using the neuroblastoma cell lineslisted in Table 2. Cells were grown to 70-80% confluence in completemedia [RPMI 1640 containing 10% FCS, 100 IU penicillin, 100 μg/mLstreptomycin and 2 mM L-glutamine], harvested, washed in complete media,and counted. Cell viability was determined by trypan blue exclusion. Thecells were placed into soft agar (0.12% in media) and plated at 5,000viable cells per well onto an agarose underlayer (0.4%) in 24-wellplates. After overnight culture, AB or Compound C was added at theindicated concentration. Control cells received media alone. As acontrol for cell death, cells were treated with a superlethal dose of 10μg/ml of cisplatin. The dosage of AB or Compound C which inhibited fiftypercent (or ninety percent) of cell proliferation (IC₅₀ or IC₉₀) wascalculated using the Chou Analysis' Median Effective Dose equation.

Clonogenicity is determined as the percentage of clones in treatedcultures relative to clones in media-treated control cultures. The IC₅₀and IC₉₀ values of AB and Compound C for the neuroblastoma cancer celllines are provided in Table 2.

The results demonstrate that Compound C is a more potent growthinhibitor than AB. The data show that Compound C and AB inhibit cellproliferation in a dose-dependent manner but that the cells are at leastan order of magnitude more sensitive to Compound C. The ratio of IC₅₀AB:IC₅₀ Compound C ranges between 6.5- to 77-fold with a median of 34μM. Similarly the ratio of IC₉₀ AB:IC₉₀ Compound C ranges between 5.8-to 342-fold with a median value of 36 μM. The IC₉₀ values are clinicallyimportant for assessing eradication of residual cancer disease.

TABLE 2 Inhibition of Established Tumor Cell Lines by AB and Compound CAB Compound C Cell Line IC₅₀ ^((a)) IC₉₀ ^((a)) IC₅₀ ^((a)) IC₉₀ ^((a))SK-N-SH 998 3397 13 29 NBAS-5 833 13030  19 38 SK-N-MC 215 1314 29 224 IMR-32 881 3566 26 44 NGP 197 1622 30 54 LA1-5S 1627  2675 21 38 SMS-KCN1872  ND 152  ND NBL-W-N 489 3074 34 78 SMS-KAN 1138  2079 27 58^((a))All concentrations are in μM.

EXAMPLE 4 Inhibition of Cancer Cell Proliferation Assessed by the SRBAssay

The inhibition of cell proliferation was measured in the cancer celllines of Table 3 using the sulforhoamine B (SRB) assay as described byMonks et al., 1991, J. Natl. Can. Inst. 83:757-766. The SRB assay isused to screen for anti-cancer drugs. A representative clonogenicitytitration curve for AB, Compound C and Compound D is shown in FIG. 1 forthe MCF-7 and CFPAC cell lines. The IC₅₀ and IC₉₀ values of AB, CompoundC and Compound D for each cancer cell line are provided in Table 3. Acomparison of Compound C, Compound D and AB demonstrates that Compound Cand Compound D at least an order of magnitude greater activity than ABas measured by the IC₅₀ and IC₉₀ values.

TABLE 3 Human Tumor Cell Lines Cell Lines Origin MCF7 Breast CarcinomaPC3 Prostate Carcinoma CFPAC Pancreatic Carcinoma HL-60 PromyelocyticLeukemia HT29 Colon Carcinoma LOXIMVI Melanoma SF-295 Glioblastoma

TABLE 4 Comparison of AB, Compound C and Compound D In The SRB Assay ABCompound C Compound D Cell IC₅₀ IC₉₀ IC₅₀ IC₉₀ IC₅₀ IC₉₀CFPAC >4.000 >4.000 0.2077 3.6386 0.1853 >4.000 HL-60 1.6438 3.45470.2048 0.2480 0.0916 0.7441 HT29 >4.000 >4.000 0.2146 0.4317 0.16370.2272 LOX IMVI >4.000 >4.000 0.1880 0.2402 0.1919 0.2451MCF7 >4.000 >4.000 0.1560 0.2332 0.0953 0.1224 PC-3 >4.000 >4.000 0.34030.4763 0.3694 0.4745 SF-295 >4.000 >4.000 0.1903 0.2425 0.1900 0.2421(a) Expressed in mM

EXAMPLE 5 Effect Compounds C, D and AN-9 on Hyperproliferating Cells

The SRB assay was used to compare the effects of Compound C, compound Dand AN-9 on the proliferation of hyperproliferating, immortalized humankeratinocytes as described in Example 4 (FIG. 2). The IC₅₀ and IC₉₀ ofCompound C on the keratinocytes was 0.050 and 0.106 mM, respectively,and the IC₅₀ and IC₉₀ of Compound D on the keratinocytes was 0.050 and0.108 mM, respectively. In contrast, the same parameters for AN-9 were0.108 and 0.221 mM, respectively.

EXAMPLE 6 Inducing Differentiation

Cancer cell differentiation was evaluated in human leukemia cell lineHL-60 by changes in expression of myelocytic maturation marker CD11b.

The level of CD11b was measured on HL-60 cells by flow cytometry using amonoclonal antibody (MAb) against CD11b in a standard indirectimmunofluorescence assay. Cells were cultured for 6 days with theindicated concentration of Compound C or AN-9. Cultured cells werecollected by centrifugation, resuspended at 10⁶ cells per 20μ RPMI+10%FCS and incubated with MAb (PE-anti-CD11b) for 60 min at 4° C. The cellswere washed twice in cold PBS+10t FCS and incubated with a 1:20 dilutionof FITC-conjugated F(ab′)² fragment of rabbit anti-mouse IgG for 20-30min at 4° C. in the dark. After washing the cells twice in cold PBS+10%FCS, the flow cytometry was performed. The results are shown in FIG. 3.Compound C was a more active differentiation inducer than was AN-9.

We claim:
 1. A method of treating a blood disorder in a subject whichcomprises administering a therapeutically-effective amount of a compoundrepresented by the Formula (I):

wherein R is C₁ to C₁₀ alkyl, C₂ to C₁₀ alkenyl or C₂ to C₁₀ alkynyl,optionally substituted with hydroxy, alkoxy, halo, trifluoromethyl, arylor heteroaryl; R¹ and R² are independently H, C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, or C₂-C₁₀ alkynyl, any of which can be optionally substitutedwith halo, alkoxy, amino, trifluoromethyl, aryl or heteroaryl; R³ is (1)C₁-C₁₂ alkyl or C₂-C₁₀ alkenyl substituted with from one to tensubstituents selected from the group consisting of hydroxy, alkoxy,acyloxy and aroyloxy, optionally having one or more halo groups, whereinadjacent hydroxy groups, if present, can optionally be present as anacetonide, (2) —(CH₂)_(n)—[O—(CH₂)_(p]) _(m)—OR⁴, (3)—(CH₂)_(n)—PEG—OR⁴, or (4) —(CH₂)_(n)—PPG—OR⁴; R⁴ is C₁ to C₆ alkyl, C₂to C₆ alkenyl, aryl, acyl, aroyl, alkoxycarbonyl, aminocarbonyl or

PEG is a polyethylene glycol moiety having an average molecular weightof 200-8000; PPG is a polypropylene glycol moiety having an averagemolecular weight of 200-8000; p is 2to 4; n is 1 to 5; m is 1 to 6; or apharmaceutically-acceptable salt thereof; with the proviso that when Ris alkyl, R³ can not be monohydroxy-n-propyl.
 2. The method of claim 1wherein R³ is —(CH₂)_(n)—[O—(CH₂)_(p)]_(m)—OR⁴, R⁴ is alkyl, p is 2 or3, n is 1 or 2, and m is 1, 2 or
 3. 3. The method of claim 1 wherein R³is —(CH₂)_(n)—(OCH₂CH₂)_(m)—OR⁴.
 4. The method of claim 1 wherein saidcompound is 2-(2-methoxy ethoxy)acetyloxymethyl butyrate,2-[2-(methoxyethoxy)ethoxy] acetyloxymethyl butyrate,1-[2-(2-methoxyethoxy)acetyl]oxyethyl butyrate, or 1-{2-[2-(2-methoxyethoxy)ethoxy]acetyl}oxyethyl butyrate.
 5. A method ofincreasing hemoglobin content in blood which comprises administering atherapeutically-effective amount of a compound of any one of claims 1 to4 to a subject.
 6. The method of claim 5 wherein said hemoglobin isfetal hemoglobin.
 7. The method of claim 6 wherein said composition isadministered orally or parenterally.
 8. The method of any one of claims1 to 4 wherein treating said blood disorder comprises increasing thehemoglobin content in cells of said subject.
 9. The method of any one ofclaims 1 to 4 wherein said disorder is selected from the groupconsisting of thalassemias, sickle cell anemias, infectious anemias,aplastic anemias, hypoplastic and hypoproliferative anemias,sideroblastic anemias, myelophthisic anemias, antibody-mediated anemias,anemias due to chronic diseases and enzyme-deficiencies, and anemias dueto blood loss, radiation therapy and chemotherapy.
 10. The method of anyone of claims 1 to 4 wherein said composition is administered orally orparenterally.
 11. A method of treating gastrointestinal disorders whichcomprises administering a therapeutically-effective amount of a compoundof any one of claims 1 to 4 to a subject.
 12. The method of claim 11wherein said compound is administered orally, parenterally,transdermally, transmucosally, intranasally, rectally or topically. 13.A method of treating cutaneous ulcers which comprises administering atherapeutically-effective amount of a compound of any one of claims 1 to4 to a subject.
 14. The method of claim 13 wherein said compound isadministered orally, parenterally, transdermally, transmucosally,intranasally, rectally or topically.
 15. A method of inducing woundhealing which comprises administering a therapeutically-effective amountof a compound of any one of claims 1 to 4 to a subject.
 16. The methodof claim 15 wherein said compound is administered orally, parenterally,transdermally, transmucosally, intrasally, rectally or topically.
 17. Amethod of treating, preventing or ameliorating symptoms ininsulin-dependent patients which comprises administering an amount of acompound of any one of claims 1 to 4 effective to enhance insulinexpression.
 18. The method of claim 17 wherein said compound isadministered orally, parenterally, transdermally, transmucosally,intranasally, rectally or topically.
 19. A method of treating,preventing or ameliorating symptoms in cystic fibrosis patients whichcomprises administering an amount of a compound of any one of claims 1to 4 effective to enhance chloride channel expression.
 20. The method ofclaim 19 wherein said compound is administered orally, parenterally,transdermally, transmucosally, intranasally, rectally or topically.